Back to EveryPatent.com
United States Patent |
6,120,765
|
Hibino
,   et al.
|
September 19, 2000
|
Urokinase plasminogen activator fragments
Abstract
Purified uPA peptides having, mitogenic activity and containing as few as
six amino acids of the EGF-like domain of uPA.
Inventors:
|
Hibino; Toshihiko (Charlestown, MA);
Takahashi; Tadahito (Charlestown, MA);
Horii; Izumi (Charlestown, MA);
Goetinck; Paul F. (Boston, MA)
|
Assignee:
|
Shiseido Co. Ltd. (JP)
|
Appl. No.:
|
142590 |
Filed:
|
October 25, 1993 |
Current U.S. Class: |
424/94.63; 435/215; 514/12; 514/13; 514/14; 514/15; 514/16; 514/17 |
Intern'l Class: |
A61K 038/49; C12N 009/72 |
Field of Search: |
424/94.63
435/215,172.3
514/12,13,14,15,16,17
|
References Cited
U.S. Patent Documents
4940666 | Jul., 1990 | Boyce et al. | 435/240.
|
4999194 | Mar., 1991 | Broeze et al. | 424/94.
|
5366862 | Nov., 1994 | Venton et al. | 435/7.
|
Other References
Appella et al., "The Receptor-binding Sequence of Urokinase", The Journal
of Biological Chemistry, vol. 262, No. 10, pp. 4437-4440, Apr. 5, 1987.
Del Rosso et al., "Modulation of Surface-Associated Urokinase: Binding,
Interiorization, Delivery to Lysosomes, and Degradation in Human
Keratinocytes", Experimental Cell Research, vol. 193, pp. 346-355, 1991.
Fibbi et al., "Interaction of Urokinase A Chain with the Receptor of Human
Keratinocytes Stimulates Release of Urokinase-like Plasminogen Activator",
Experimental Cell Research, vol. 187, pp. 33-38, 1990.
Fraki et al., "Uninvolved Skin from Psoriatic Patients Develops Signs of
Involved Psoriatic Skin After Being Grafted onto Nude Mice", Science, vol.
215, pp. 685-687, Feb. 5, 1982.
Grant et al., "Active and Pro-plasminogen Activator on the Surface of Human
Bladder Cancer Cells Derived from a High Grade Invasive Tumor",
Biochemical and Biophysical Research Communications, vol. 172, No. 2, pp.
870-876, Oct. 30, 1990.
Gr.o slashed.ndahl-Hansen et al., "Urokinase-and Tissue-Type Plasminogen
Activators in Keratinocytes During Wound Reepithelialization In Vivo",
Invest. Dermatol., vol. 90, No. 6, pp. 790-795, Jun. 1988.
Hibino et al., "Enhanced Expression of Human Pro-urokinase cDNA in
Escherichia coli", Agric. Biol. Chem., vol. 52, No. 2, pp. 329-336, 1988.
Kirchheimer et al., "Proliferation of a human epidermal tumor cell line
stimulated by urokinase", FASEB J., vol. 1, No. 2, pp. 125-128, 1987.
Lazarus et al., "Proteinase Metabolism in Human Skin: The Role of
Plasminogene Activator and Mast Cell Proteinases in Cutaneous Biology",
Physiology, Biochemistry, and Molecular Biology of the Skin, 2.sup.ed,
Editor Goldsmith, 1991.
Meissauer et al., "Urokinase-Type and Tissue-Type Plasminogen Activators
are Essential for in Vitro Invasion of Human Melanoma Cells", Experimental
Cell Research, vol. 192, pp. 453-459, 1991.
Miganatti et al., "Tumor Invasion through the Human Amniotic Membrane
Requirement for a Proteinase cascade", Cell, vol. 47, pp. 487-498, Nov.
21, 1986.
Nielsen et al., "A 55,000-60,000 M.sub.r Receptor Protein for
Urokinase-type Plasminogen Activator", The Journal of Biological
Chemistry, vol. 263, No. 5, pp. 2358-2363, Feb. 15, 1988.
Nykjaer et al., "Urokinase receptors in human monocytes", Biochimica et
Biophysica Acta, vol. 1050, pp. 399-407, 1990.
Rabbani et al., "An Amino-Terminal Fragment of Urokinase Isolated from a
Prostate Cancer Cell Line (PC-3) is Mitogenic for Osteoblast-like Cells",
Biochemical and Biophysical Research Communications, vol. 173, No. 3, pp.
1058-1064, Dec. 31, 1990.
Riccio et al., "The human urokinase-plasminogen activator gene and its
promoter", Nucleic Acids Research, vol. 13, No. 8, pp. 20-30, 1985.
Roldan et al., "Cloning and expression of the receptor for human urokinase
plasminogen activator, a central molecule in cell surface, plasmin
dependent proteolysis", The EMBO Journal, vol. 9, No. 2, pp. 467-474,
1990.
Tsubol et al., "Bimodal Relationship Between Invasion of the Amniotic
Membrane and Plasminogen Activator Activity", Int. J. Cancer, vol. 46, pp.
56-60, 1990.
Fry et al., "Observations on Mitosis in Psoriatic Epidermis", British
Journal Dermatology, vol. 82, pp. 19-22, 1970.
Hibino et al., "Urokinase-Plasmonogen Activator (u-PA) is Associated with
Keratinocyte Proliferation", XVII MGH Research Symposium and Poster
Session, Abstracts of Posters and Oral Presentations, Abst No. 158, Jan.
20, 1993.
Wun et al., "A Proenzyme Form of Human Urokinase", Journal Biological
Chemistry, vol. 257, No. 12, pp. 7262-7268, 1982.
Hibino et al., "EGF-like domain of urokinase plasmogen-activator (uPA)
stimulates keratinocyte growth", The American Society for Cell Biology,
Abstract presented at the Molecular Biology of the Cell 4(Suppl.) 1993.
2/A.
Del Rosso, et al., "Role of specific membrane receptors in urokinase
dependent migration of human keratinocytes", The Journal of Investigative
Dermatology, vol. 94, No. 3, pp. 310-316, Mar. 1990.
Hibino et al., "EGF-like domain of urokinase plasmogen activator UPA plays
an essential role in keratinocyte growth stimulation" Journal of
Investigative Dermatology, 100(4):500 (1993).
Rabbani, S.A. et al. J. Biol. Chem. 267:14151-14156 (1992).
Au et al. Nucleotide and deduced amino acid sequences of baboon
urokinase-type plasminogen activator. Nucleic Acids Res. (1990) 18:3411.
Sambrook et al. Molecular Cloning: A Laboratory Manual Second Edition vols.
1, 2 and 3. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New
York, U.S.A. Nov., 1989.
Mather et al. The major fat-globule membrane proteins, bovine components
15/16 and guinea-pig GP 55, are homologous to MGF-E8 a murine glycoprotein
containing epidermal growth factor-like and factor V/VIII-like sequences.
Biochem. Mol. Biol. Int., (199, Mar. 1993.
|
Primary Examiner: Kemmerer; Elizabeth
Assistant Examiner: Romeo; David S.
Attorney, Agent or Firm: Fish & Richardson P.C.
Parent Case Text
This application is a continuation-in-part of U.S. Ser. No. 08/042,318,
filed on Apr. 2, 1993 now abandoned , which is hereby incorporated by
reference.
Claims
What is claimed is:
1. A purified peptide containing more than 5 and less than 13 contiguous
amino acid residues from the 18 kilodalton amino terminal region of
urokinase plasminogen activator, wherein said amino acid residues include
the amino acid sequence Ser-Asn-Lys-Tyr-Phe-Ser (SEQ ID NO: 2).
2. The purified peptide of claim 1, including the amino acid sequence
Thr-Cys-Val-Ser-Asn-Lys-Tyr-Phe-Ser-Asp-Ile-His (SEQ ID NO: 6).
3. The purified peptide of claim 1 wherein said peptide contains more than
5 and less than 10 contiguous amino acid residues.
4. The purified peptide of claim 3, wherein said contiguous amino acid
residues include the amino sequence Ser-Asn-Lys-Tyr-Phe-Ser-Asn-Ile-His
(SEQ ID NO: 1).
5. The purified peptic of claim 1, wherein said peptide contains 6
contiguous amino acid residues of said 18 kilodalton amino terminal
region.
6. A therapeutic composition comprising as an active ingredient a peptide
of claim 1 and a pharmaceutically-acceptable carrier.
7. A peptide produced by expression of isolated DNA comprising a sequence
encoding a peptide of claim 1, which is less than 13 amino acid residues
in length.
8. The purified peptic of claim 1, wherein the purified peptide is
Ser-Asn-Lys-Tyr-Phe-Ser (SEQ ID NO: 2).
9. The purified peptide of claim 1, wherein the purified peptide is
Thr-Cys-Val-Ser-Asn-Lys-Tyr-Phe-Ser-Asp-Ile-His (SEQ ID NO: 6).
10. The purified-peptide of claim 1, wherein the purified peptide is
Ser-Asn-Lys-Tyr-Phe-Ser-Asn-lle-His (SEQ ID NO: 1).
11. The purified peptide of claim 1, containing more than 5 and less than
12 contiguous amino acid residues from the 18 kilodalton amino terminal
region of urokinase plasminogen activator.
12. The purified peptide of claim 1, wherein said peptide contains more
than 5 and less than 10 contiguous amino acid residues, inclusive of said
18 kilodalton amino terminal region of urokinase plasminogen activator.
13. A purified peptide consisting of the amino acid sequence
Asn-Gly-Gly-Thr-Cys-Val-Ser-Asn-Lys-Tyr-Phe-Ser-Asp-Ile-His-Trp-Cys-Asn
(SEQ ID NO:4).
Description
BACKGROUND OF THE INVENTION
The invention relates generally to urokinase plasminogen activator and
fragments thereof.
Two types of plasminogen activators, tissue type plasminogen activator
(tPA) and urokinase, or urokinase plasminogen activator (uPA), are known.
tPA binds directly to fibrin clots where it activates the conversion of
plasminogen to plasmin. uPA is a serine protease with systemic activity.
It binds to the receptor found on many cell types and converts plasminogen
to plasmin on the cell surface.
SUMMARY OF THE INVENTION
uPA is an approximately 55 Kd molecule which consists of (beginning at the
N-terminal end) an EGF-like domain (EGF) (which corresponds to residues
1-45) a kringle domain (which corresponds to residues 46-157), and a
trypsin-like protease domain (which corresponds to residues 158-411). The
EGF and kringle domains make up the amino terminal fragment (AFF (SEQ ID
NO:25)), which is mitogenic for human keratinocytes. uPA binds, by its
EGF-like domain, to a specific membrane receptor (uPAR) expressed in many
cell types. The EGF-like domain is often referred to as the growth factor
domain (GFD). The amino acid residue numbering system used herein begins
with residue 1 at the N terminal end of uPA.
The inventors have discovered that peptides containing as few as six amino
acids of the EGF-like domain of uPA have mitogenic activity and are
thought to bind to the urokinase plasminogen activator receptor (uPAR).
In general, the invention features, a uPA peptide, preferably a purified
peptide, (which is capable of either or both of binding to or inducing
mitogenesis in cells bearing the uPAR, e.g., epidermal cells, e.g.,
keratinocytes) which consists essentially of or contains more than 5 and
less than 15 (or more preferably more than 5 and less than 19), contiguous
amino acid residues from the growth factor domain, e.g., residues 16
through 33 (SEQ ID NO:23) (or more preferably 14 through 33), of urokinase
plasminogen activator.
In preferred embodiments the peptide contains more than 5 and less than 13,
(or more preferably more than 5 and less than 16), contiguous amino acid
residues, inclusive, of the growth factor domain; the peptide includes the
amino acid sequence Thr-Cys-Val-Ser-Asn-Lys-Tyr-Phe-Ser-Asp-Ile-His (SEQ
ID NO:6); the peptide contains more than 5 and less than 10 contiguous
amino acid residues, inclusive, of the domain growth factor; the peptide
includes the amino acid sequence Ser-Asn-Lys-Tyr-Phe-Ser-Asp-Ile-His (SEQ
ID NO:1); the peptide contains 6 contiguous amino acid residues of the
domain growth factor; the peptide includes the amino acid sequence
Ser-Asn-Lys-Tyr-Phe-Ser (SEQ ID NO:2).
In preferred embodiments the peptide is mitogenic for cells bearing the
uPAR; the peptide binds to the uPAR but is not mitogenic for cells bearing
the uPAR.
In another aspect, the invention includes a uPA peptide, preferably a
purified peptide, (which is capable of either or both of binding to or
inducing mitogenesis in cells bearing the uPAR, e.g., epidermal cells,
e.g., keratinocytes) which consists essentially of or contains more than 5
and less than 12 contiguous amino acid residues from the amino terminal
fragment, e.g., from the growth factor domain, e.g., from residues 15
through 30 of uPA.
In preferred embodiments the peptide contains more than 5 and less than 10
contiguous amino acid residues, inclusive, of the amino terminal fragment
of urokinase plasminogen activator; the peptide includes the amino acid
sequence Ser-Asn-Lys-Tyr-Phe-Ser-Asp-Ile-His (SEQ ID NO:1); the peptide
contains 6 contiguous amino acid residues, inclusive, of the amino
terminal fragment of urokinase plasminogen activator; the peptide includes
the amino acid sequence Ser-Asn-Lys-Tyr-Phe-Ser (SEQ ID NO:2).
In preferred embodiments the peptide is mitogenic for cells bearing the
uPAR; the peptide binds to the uPAR but is not mitogenic for cells bearing
the uPAR.
In another aspect the invention includes the peptide, preferably in a
purified preparation,
Asn-Gly-Gly-Thr-Cys-Val-Ser-Asn-Lys-Tyr-Phe-Ser-Asn-Ile-His-Trp-Cys-Asn
(SEQ ID NO:4).
In another aspect, the invention features a uPA, preferably a purified
peptide, peptide in which the amino acid residue at position 23 is an
amino acid other than L-lysine. In preferred embodiments: the amino acid
residue at position 23 is an amino acid having a side chain with a
net-positive charge at a physiological pH, e.g., at pH 7; amino acid
residue at position 23 is an amino acid having a side chain which is more
positively charged than is the side chain of lysine; the amino acid
residue at position 23 is a basic amino acid; the amino acid residue at
position 23 is any of arginine, histidine, or a positively charged or
basic non-naturally occurring amino acid.
In other preferred embodiments: the amino acid residue at position 23 is an
amino acid having a side chain with a net-negative charge at a
physiological pH, e.g., at pH 7; the amino acid residue at position 23 is
an amino acid having a side chain which is more negatively charged than is
the side chain of lysine; the amino acid residue at position 23 is an
acidic amino acid; the amino acid residue at position 23 is any of
aspartic acid, glutamic acid, or a negatively charged or acidic
non-naturally occurring amino acid.
In preferred embodiments the peptide is a full length uPA peptide.
In another aspect, the invention features a uPA peptide, preferably a
purified peptide, which includes a sequence of the formula: n-R.sup.2
-Asn-R.sup.1 -Tyr-Phe-R.sup.3 -c, wherein,
R.sup.1 is an amino acid residue other than L-lysine;
R.sup.2 is a sequence of between 1 and 21 residues in length, having as its
carboxy-terminus Ser.sup.21 of uPA and extending, inclusive of Ser.sup.21,
from between 1 and 21 amino acid residues in the N-terminal direction of
uPA; and
R.sup.3 is a sequence of between 1 and 25 residues in length having as its
amino-terminus Ser.sup.26 of uPA and extending, inclusive of Ser.sup.26,
from between 1 and 25 amino acid residues in the C-terminal direction of
uPA;
wherein c indicates the carboxy terminal direction of the peptide and n
indicates the amino terminal direction of the peptide.
In preferred embodiments: R.sup.1 is an amino acid having a side chain with
a net-positive charge at a physiological pH, e.g., at pH 7; R.sup.1 is a
basic amino acid; R.sup.1 is any of arginine, histidine, or a positively
charged or basic non-naturally occurring amino acid.
In preferred embodiments: R.sup.2 is any of:
n-Asp-Cys-Leu-Asn-Gly-Gly-Thr-Cys-Val-Ser-c (SEQ ID NO:9);
n-Cys-Leu-Asn-Gly-Gly-Thr-Cys-Val-Ser-c; (SEQ ID NO:10)
n-Leu-Asn-Gly-Gly-Thr-Cys-Val-Ser-c; (SEQ ID NO:11)
n-Asn-Gly-Gly-Thr-Cys-Val-Ser-c (SEQ ID NO:12);
n-Gly-Gly-Thr-Cys-Val-Ser-c (SEQ ID NO:13);
n-Gly-Thr-Cys-Val-Ser-c (SEQ ID NO:14);
n-Thr-Cys-Val-Ser-c (SEQ ID NO:15);
n-Cys-Val-Ser-c;
n-Val-Ser-c; or
n-Ser-c
In preferred embodiments: R.sup.3 is any of:
n-Ser-c;
n-Ser-Asn-c;
n-Ser-Asn-Ile-c;
n-Ser-Asn-Ile-His-c (SEQ ID NO:16);
n-Ser-Asn-Ile-His-Trp-c (SEQ ID NO:17);
n-Ser-Asn-Ile-His-Trp-Cys-c (SEQ ID NO:18); or
n-Ser-Asn-Ile-His-Trp-Cys-Asn-c (SEQ ID NO:19).
In preferred embodiments:
R.sup.2 is n-Asp-Cys-Leu-Asn-Gly-Gly-Thr-Cys-Val-Ser-c (SEQ ID NO:9) and
R.sup.3 is n-Ser-Asn-Ile-His-Trp-Cys-Asn-c (SEQ ID NO:19);
R.sup.2 is n-Asn-Gly-Gly-Thr-Cys-Val-Ser-c (SEQ ID NO:2); and R.sup.3 is
n-Ser-Asn-Ile-His-Trp-Cys-Asn-c (SEQ ID NO:19);
R.sup.2 is n-Thr-Cys-Val-Ser-c (SEQ ID NO:15); and R.sup.3 is
n-Ser-Asn-Ile-His-Trp-Cys-Asn-c (SEQ ID NO:19);
R.sup.2 is n-Thr-Cys-Val-Ser-c (SEQ ID NO:15); and R.sup.3 is
n-Ser-Asn-Ile-His-c; (SEQ ID NO:16)
R.sup.2 is n-Ser-c and R.sup.3 is n-Ser-Asn-Ile-His-c (SEQ ID NO:16); or
R.sup.2 is n-Ser-c and R.sup.3 is n-Ser-c; R.sup.1 is Arg.
In another aspect, the invention features a uPA peptide, preferably a
purified peptide, including a sequence of the formula: n-R.sup.2
-Asn-R.sup.1 -Tyr-Phe-R.sup.3 -c, wherein,
R.sup.1 is an amino acid residue other than L-lysine;
R.sup.2 is a sequence of between 1 and 21 residues in length, having as its
carboxy-terminus Ser.sup.21 of uPA and extending, inclusive of Ser.sup.21,
from between 1 and 21 amino acid residues in the N-terminal direction of
uPA; and
R.sup.3 is a sequence of between 1 and 25 residues in length having as its
amino-terminus Ser.sup.26 of uPA and extending, inclusive of Ser.sup.26,
from between 1 and 25 amino acid residues in the C-terminal direction of
uPA;
wherein c indicates the carboxy terminal direction of the peptide and n
indicates the amino terminal direction of the peptide.
In preferred embodiments: R.sup.1 is an amino acid having a side chain with
a net-negative charge at a physiological pH, e.g., at pH 7; R.sup.1 is an
amino acid having a side chain which is more negatively charged than is
the side chain of lysine; R.sup.1 is an acidic amino acid; R.sup.1 is any
of aspartic acid, glutamic acid, or a negatively charged or acidic
non-naturally occurring amino acid.
In preferred embodiments: R.sup.2 is any of:
n-Asp-Cys-Leu-Asn-Gly-Gly-Thr-Cys-Val-Ser-c (SEQ ID NO:9);
n-Cys-Leu-Asn-Gly-Gly-Thr-Cys-Val-Ser-c (SEQ ID NO:10);
n-Leu-Asn-Gly-Gly-Thr-Cys-Val-Ser-c (SEQ ID NO:11);
n-Asn-Gly-Gly-Thr-Cys-Val-Ser-c (SEQ ID NO:12);
n-Gly-Gly-Thr-Cys-Val-Ser-c (SEQ ID NO:13);
n-Gly-Thr-Cys-Val-Ser-c (SEQ ID NO:14);
n-Thr-Cys-Val-Ser-c (SEQ ID NO:15);
n-Cys-Val-Ser-c;
n-Val-Ser-c; or
n-Ser-c
In preferred embodiments: R.sup.3 is any of:
n-Ser-c;
n-Ser-Asn-c;
n-Ser-Asn-Ile-c;
n-Ser-Asn-Ile-His-c (SEQ ID NO:16);
n-Ser-Asn-Ile-His-Trp-c (SEQ ID NO:17);
n-Ser-Asn-Ile-His-Trp-Cys-c (SEQ ID NO:18); or
n-Ser-Asn-Ile-His-Trp-Cys-Asn-c (SEQ ID NO:19).
In preferred embodiments:
R.sup.2 is n-Asp-Cys-Leu-Asn-Gly-Gly-Thr-Cys-Val-Ser-c (SEQ ID NO:9) and
R.sup.3 is n-Ser-Asn-Ile-His-Trp-Cys-Asn-c (SEQ ID NO:19);
R.sup.2 is n-Asn-Gly-Gly-Thr-Cys-Val-Ser-c (SEQ ID NO:12); and R.sup.3 is
n-Ser-Asn-Ile-His-Trp-Cys-Asn-c (SEQ ID NO:19);
R.sup.2 is n-Thr-Cys-Val-Ser- (SEQ ID NO:15)c; and R.sup.3 is
n-Ser-Asn-Ile-His-Trp-Cys-Asn-c (SEQ ID NO:19);
R.sup.2 is n-Thr-Cys-Val-Ser-c (SEQ ID NO:15); and R.sup.3 is
n-Ser-Asn-Ile-His-c (SEQ ID NO:16);
R.sup.2 is n-Ser-c and R.sup.3 is n-Ser-Asn-Ile-His-c (SEQ ID NO:16); or
R.sup.2 is n-Ser-c and R.sup.3 is n-Ser-c;
R.sup.1 is glutamic acid.
In another aspect, the invention features a therapeutic composition
including a uPA peptide of the invention and a pharmaceutically-acceptable
carrier.
In preferred embodiments: the peptide is mitogenic for cells bearing the
uPAR; the peptide inhibits mitogenic activity in cells bearing the uPAR;
the peptide binds to the uPAR but is not mitogenic for cells bearing the
uPAR.
In another aspect, the invention features a method of regulating, e.g.,
promoting or inhibiting, the growth or proliferation of a cell, e.g., a
cell expressing the urokinase plasminogen activator receptor, e.g., an
epidermal cell, e.g., a keratinocyte, or an osteoblast. The method
includes administering to the cell a growth regulating amount of a uPA
peptide the invention.
In preferred embodiments: the peptide is mitogenic for cells bearing the
uPAR; the peptide inhibits mitogenic activity in cells bearing the uPAR;
the peptide binds to the uPAR but is not mitogenic for cells bearing the
UPAR.
In preferred embodiments, the uPA peptide promotes cell growth or
proliferation and the method further includes administering a growth
promoting compound, other than a uPA peptide. The compound can be, e.g., a
peptide growth factor, e.g., epidermal growth factor or insulin, a complex
mixture or extract, e.g., pituitary extract, or a non-peptide compound,
e.g., hydrocortisone.
In another aspect, the invention features, a method of regulating, e.g.,
promoting or inhibiting, the growth or proliferation of epidermal tissue
in a patient which has been subjected to trauma, e.g., trauma arising from
a disease, e.g., a disease producing an ulceration of epidermal tissue, a
surgical incision, a wound, e.g., from a mechanical injury, e.g., a cut,
or a burn. The method includes administering to the tissue a growth
regulating amount of a uPA peptide of the invention.
In preferred embodiments: the peptide is mitogenic for cells bearing the
uPAR; the peptide inhibits mitogenic activity in cells bearing the uPAR;
the peptide binds to the uPAR but is not mitogenic for cells bearing the
uPAR.
In preferred embodiments, the uPA peptide promotes cell growth or
proliferation and the method further includes administering a growth
promoting compound, other than a uPA peptide. The compound can be, e.g., a
peptide growth factor, e.g., epidermal growth factor or insulin, a complex
mixture or extract, e.g., pituitary extract, or a non-peptide compound,
e.g., hydrocortisone.
In another aspect, the invention features a method of regulating, e.g.,
promoting or inhibiting, the growth of cells, e.g., a sheet of cells,
e.g., a sheet of epidermal cells, e.g., keratinocytes, in vitro. The
method includes culturing the cells in the presence of an effective amount
of a uPA peptide of the invention.
In preferred embodiments: the peptide is mitogenic for cells bearing the
uPAR; the peptide inhibits mitogenic activity in cells bearing the uPAR;
the peptide binds to the uPAR but is not mitogenic for cells bearing the
uPAR.
In preferred embodiments, the uPA peptide promotes cell growth or
proliferation and the method further includes administering a growth
promoting compound, other than a uPA peptide. The compound can be, e.g., a
peptide growth factor, e.g., epidermal growth factor or insulin, a complex
mixture or extract, e.g., pituitary extract, or a non-peptide compound,
e.g., hydrocortisone.
In another aspect, the invention features, a method for treating an area of
denuded skin in a patient, arising, e.g., from a burn, a wound, or a
surgical procedure. The method includes applying cells, e.g., epidermal
cells, e.g., a sheet of epidermal cells, produced according to a method of
the invention to allow effective attachment of the cells to the underlying
dermis of the patient. The method can include administering a uPA peptide,
e.g., a growth promoting peptide of the invention, to the patient before
or after the sheet is applied to the patient.
In preferred embodiments, the uPA peptide promotes cell growth or
proliferation and the method further includes administering a growth
promoting compound, other than a uPA peptide. The compound can be, e.g., a
peptide growth factor, e.g., epidermal growth factor or insulin, a complex
mixture or extract, e.g., pituitary extract, or a non-peptide compound,
e.g., hydrocortisone.
In another aspect, the invention includes, a method of identifying an
antagonist of uPA, e.g., of the mitogenic activity of urokinase
plasminogen activator. The method includes: culturing cells, e.g., uPAR
bearing cells, e.g., epidermal cells, in the presence of a uPA peptide of
the invention; contacting the cells with a candidate compound; and
comparing the level of mitogenic activity in the presence of the candidate
compound to the level of mitogenic activity in the absence of the
candidate compound, a lower level of activity in the presence of the
compound being indicative that the compound is an antagonist. The
candidate compound can be, e.g., an antibody, preferably a monoclonal
antibody, e.g., an antibody to the uPAR, or a peptide, Mitogenic activity
can be measured, e.g., by the level of [.sup.3 H]-thymidine incorporation.
In another aspect, the invention includes, a method of identifying an
agonist of uPA, e.g., of the mitogenic activity of urokinase plasminogen
activator. The method includes: culturing cells, e.g., uPAR bearing cells,
e.g., epidermal cells, in the presence of a uPA peptide, e.g., a uPA
peptide which inhibits mitiogenic activity, contacting the cells with a
candidate compound; and comparing the level of mitogenic activity, in the
presence of the candidate compound to the level of mitogenic activity in
the absence of the candidate compound, a higher level of activity in the
presence of the compound being indicative that the compound is an agonist.
In another aspect, the invention features, a method of inhibiting the
interaction of urokinase plasminogen activator with urokinase plasminogen
activator receptor. The method includes contacting the receptor with a uPA
peptide of the invention to inhibit the interaction.
In preferred embodiments: the peptide is mitogenic for cells bearing the
uPAR; the peptide inhibits mitogenic activity in cells bearing the uPAR;
the peptide binds to the uPAR but is not mitogenic for cells bearing the
uPAR.
In another aspect, the invention features, a method of inhibiting the
binding of urokinase plasminogen activator to a cell which expresses uPAR.
The method includes contacting the cell with a uPA peptide of the
invention to inhibit the interaction.
In preferred embodiments: the peptide is mitogenic for cells bearing the
uPAR; the peptide inhibits mitogenic activity in cells bearing the uPAR;
the peptide binds to the uPAR but is not mitogenic for cells bearing the
uPAR.
In another aspect, the invention features, a method of determining the
growth stage of a cell, e.g., a keratinocyte, including determining the
level (e.g., by the use of an antibody or a nucleic acid probe) of
urokinase plasminogen activator receptor expressed by the cell.
In another aspect, the invention features, a method for treating an animal
having a disorder, e.g., a disorder characterized by an unwanted
proliferation of cells, e.g., an unwanted proliferation of uPAR-bearing
cells, e.g., an epidermal disorder, e.g., psoriasis, or cancer. The method
includes: identifying an animal, e.g., a human, at risk for the disorder;
and administering a therapeutically-effective amount of a uPA peptide of
the invention to the animal.
In preferred embodiments: the peptide inhibits mitogenesis of uPAR bearing
cells; the peptide is mitogenic for cells bearing the uPAR; the peptide
binds to the uPAR but is not mitogenic for cells bearing the uPAR.
In preferred embodiments, the uPA peptide promotes cell growth or
proliferation and the method further includes administering a growth
promoting compound, other than a uPA peptide. The compound can be, e.g., a
peptide growth factor, e.g., epidermal growth factor or insulin, a complex
mixture or extract, e.g., pituitary extract, or a non-peptide compound,
e.g., hydrocortisone.
In another aspect, the invention features, a method for mitogenically
stimulating a cell bearing a uPAR, e.g., a keratinocytic cell, including
contacting the cell with an effective amount of a uPA peptide of the
invention e.g., a fragment of the AFF of uPA having at least six
contiguous residues from the GFD.
In another aspect, the invention features, a method for inhibiting
mitogenesis in a cell bearing the uPAR, e.g., a keratinocytic cell,
including contacting the cell with an effective amount of a uPA peptide of
the invention e.g., a fragment of the AFF of uPA having at least six
contiguous residues from the GFD.
In another aspect, the invention features, a method of inhibiting the
interaction of urokinase plasminogen activator with urokinase plasminogen
activator receptor on a keratinocyte including contacting the receptor
with an antibody to the urokinase plasminogen activator receptor to
inhibit the interaction.
In another aspect, the invention features, a method for inhibiting the
growth or proliferation of a cell bearing a uPAR, e.g., a dermal cell,
e.g., a keratinocytic cell including contacting the cell with an antibody
to the urokinase plasminogen activator receptor to inhibit growth.
In another aspect, the invention features a method of delivering a
compound, e.g., a toxin molecule, e.g., a peptide toxin, to a uPAR bearing
cell including providing a chimeric molecule which includes the compound
coupled, e.g., by a covalent bond, e.g., by a peptide bond, to a fragment
of the AFF of uPA at least six residues in length, e.g., to a uPA peptide
disclosed herein.
In another aspect, the invention features, a method of inhibiting the
proteolytic destruction of an extracellular protein matrix which includes
cells bearing the uPAR, e.g., basement membrane, including contacting
cells bearing the uPAR with a uPA peptide of the invention.
In preferred embodiments, the uPA peptide promotes cell growth or
proliferation and the method further includes administering a growth
promoting compound, other than a uPA peptide. The compound can be, e.g., a
peptide growth factor, e.g., epidermal growth factor or insulin, a complex
mixture or extract, e.g., pituitary extract, or a non-peptide compound,
e.g., hydrocortisone.
In preferred embodiments: the peptide inhibits mitogenesis in uPAR bearing
cells; the peptide is mitogenic for cells bearing the uPAR; the peptide
binds to the uPAR but is not mitogenic for cells bearing the uPAR.
In another aspect, the invention features, a purified DNA comprising a
sequence encoding a uPA peptide of the invention; a vector including a DNA
sequence encoding a peptide of the invention; a cell containing the
purified DNA, e.g., a cell capable of expressing peptide; an essentially
homogeneous population of cells, each of which comprises the isolated DNA;
a recombinantly produced peptide of the invention; and a method for
manufacture of a peptide of the invention including culturing the cell in
a medium to express a peptide of the invention.
The inventors have also discovered that lysine, and analogs of lysine,
stimulate mitogenisis of uPAR bearing cells. Accordingly, in another
aspect, the invention features a method of regulating growth of a cell,
e.g., a uPAR bearing cell, e.g., a keratinocyte in vitro, or in vivo,
including contacting the cell with, e.g., topically administering to the
cell, a growth regulating amount of lysine, or an analog of lysine, e.g.,
epsilon-amino caproic acid or tranexamic acid (trans-4-(amino methyl)
cyclohexane carboxylic acid.
In preferred embodiments: the lysine or analog thereof is provided at a
concentration greater than is provided in growth medium used to culture
said cells; lysine is provided at a concentration greater than found in
fetal calf or bovine serum.
In other preferred embodiments: the cell is an animal cell, e.g., a human
cell, e.g., a keratinocyte the lysine or analog thereof is contacted with
the cell in situ, i.e., when the cell is part of the animal and: the
concentration of lysine or an analog thereof at the surface of the cell is
greater than the highest concentration that can be achieved at the surface
of the cell by oral, intravenous, or other systemic administration of
lysine or an analog thereof without deleterious effect on the animal; the
concentration of lysine or an analog at the surface of the cell is greater
than the highest concentration that is achieved at the surface of the cell
when lysine or an analog thereof is administered by oral, intravenous, or
other systemic administration for nutritional purposes; the lysine or
analog thereof is administered to the animal at a concentration amount or
dosage higher than the highest concentration, amount, or dosage that can
be administered by oral, intravenous, or other systemic administration
without deleterious effect on the animal; the lysine or analog thereof is
administered to the animal at a concentration, amount, or dosage higher
than the highest concentration, amount, or dosage that is administered by
oral, intravenous, or other systemic administration of lysine or an analog
thereof for nutritional purposes; sufficient lysine is administered such
that the concentration of lysine in the dermis is higher, e.g., at least
20, 50, 80, 200, or 400% higher than the concentration of lysine in the
dermis of a normal individual or of an individual receiving intravenous
nutrition.
In another aspect, the invention features a method of regulating growth of
epidermal tissue in a patient which has been subjected to trauma including
administering, e.g., topically administering, e.g., to the traumatized
tissue, a growth regulating amount of lysine, or an analog of lysine,
e.g., epsilon-amino caproic acid or tranexamic acid (trans-4-(amino
methyl) cyclohexane carboxylic acid.
In preferred embodiments: the concentration of lysine or an analog thereof
at the surface of a cell in the treated tissue is greater than the highest
concentration that can be achieved at the surface of a cell in the treated
tissue by oral, intravenous, or other systemic administration of lysine or
an analog thereof without deleterious effect on the animal; the
concentration of lysine or an analog the surface of a cell in the treated
tissue is greater than the highest concentration that is achieved at the
surface of a cell in the treated tissue when lysine or an analog thereof
is administered by oral, intravenous, or other systemic administration for
nutritional purposes; the lysine or analog thereof is administered to the
animal at a concentration, amount, or dosage higher than the highest
concentration, amount, or dosage that can be administered by oral,
intravenous, or other systemic administration without deleterious effect
on the animal; the lysine or analog thereof is administered to the animal
at a concentration, amount, or dosage higher than the highest
concentration, amount, or dosage that is administered by oral,
intravenous, or other systemic administration for nutritional purposes;
sufficient lysine is administered such that the concentration of lysine in
the dermis is higher, e.g., at least 20, 50, 80, 200, or 400% higher than
the concentration of lysine in the dermis of a normal individual or of an
individual receiving intravenous nutrition.
In another aspect, the invention features a method of regulating the growth
of cells, e.g., uPAR bearing cells, e.g., epidermal cells, e.g.,
keratinocytes, in vitro including culturing the cells in the presence of
lysine, or an analog of lysine, e.g., epsilon-amino caproic acid or
tranexamic acid (trans-4-(amino methyl) cyclohexane carboxylic acid. In
preferred embodiments the lysine or analog thereof is provided at a higher
concentration than is lysine or an analog thereof in media used to
culture, wash, or otherwise treat the cells; lysine is present in greater
concentration than is found in fetal or bovine calf serum.
In another aspect, the invention features a method for treating an area of
denuded skin in a patient comprising applying cells, e.g., uPAR bearing
cells, e.g., epidermal cells, e.g., keratinocytes, produced according to a
method described herein to allow effective attachment of the cells to the
underlying dermis.
In preferred embodiments: the method further includes contacting lysine or
an analog thereof with the cells after they have been applied to the
patient; the concentration of lysine or an analog at the surface of the
cells is greater than the highest concentration that can be achieved at
the surface of the cells by oral, intravenous, or other systemic
administration of lysine or an analog thereof without deleterious effect
on the patient; the concentration of lysine or analog thereof at the
surface of the cells is greater than the highest concentration that is
achieved at the surface of the cells when lysine or an analog thereof is
administered by oral, intravenous, or other systemic administration of
lysine or an analog thereof for nutritional purposes; the lysine or analog
thereof is administered to the patient at a concentration, amount, or
dosage higher than the highest concentration amount or dosage that can be
administered by oral, intravenous, or other systemic administration
without deleterious effect on the patient; the lysine or analog thereof is
administered to the patient at a concentration, amount, or dosage higher
than the highest concentration, amount, or dosage that is administered by
oral, intravenous, or other systemic administration for nutritional
purposes; sufficient lysine is administered such that the concentration of
lysine in the dermis is higher, e.g., at least 20, 50, 80, 200, or 400%
higher than the concentration of lysine in the dermis of a normal
individual or of an individual receiving intravenous nutrition.
In another aspect, the invention features, a method of promoting the growth
of a cell, e.g., cell bearing a uPAR receptor, e.g., a dermal cell, e.g.,
a keratinocyte, which has been transferred to a site, e.g., the site of a
disorder, e.g., a skin disorder or a wound, on a patient including
providing the cell, applying the cell to the site, and administering to
the patient, e.g., by topical application to the site, a growth promoting
amount of lysine or an analog of lysine, e.g., epsilon-amino caproic acid
or tranexamic acid (trans-4-(amino methyl) cyclohexane carboxylic acid.
In preferred embodiments: the lysine or analog thereof is provided at a
concentration greater than is provided in growth medium used to culture
said cells; lysine is provided at a concentration greater than found in
fetal calf or bovine serum.
In other preferred embodiments: the concentration of lysine or an analog
thereof at the surface of the cell is greater than the highest
concentration that can be achieved at the surface of the cell by oral,
intravenous, or other systemic administration without deleterious effect
on the patient; the concentration of lysine an analog thereof at the
surface of the cell is greater than the highest concentration that is
achieved at the surface of the cell when lysine an analog thereof is
administered by oral, intravenous, or other systemic administration for
nutritional purposes; the lysine is administered to the patient at a
concentration, amount, or dosage higher than the highest concentration,
amount, or dosage that can be administered by oral, intravenous, or other
systemic administration without deleterious effect on the patient; the
lysine an analog thereof is administered to the patient at a
concentration, amount, or dosage higher than the highest concentration,
amount, or dosage that is administered by oral, intravenous, or other
systemic administration for nutritional purposes; sufficient lysine is
administered such that the concentration of lysine in the dermis is
higher, e.g., at least 20, 50, 80, 200, or 400% higher than the
concentration of lysine in the dermis of a normal individual or of an
individual receiving intravenous nutrition.
In another aspect, the invention features a method of identifying an
antagonist of the mitogenic activity of urokinase plasminogen activator
including culturing uPAR bearing cells in the presence of lysine, or an
analog of lysine, e.g., epsilon-amino caproic acid or tranexamic acid
(trans-4-(amino methyl) cyclohexane carboxylic acid, contacting said cells
with a candidate compound, and comparing the level of mitogenic activity
in the presence of the candidate compound to the level of mitogenic
activity in the absence of the candidate compound, a lower level of said
activity in the presence of the compound being indicative that the
compound is an antagonist.
In another aspect, the invention features a method identifying an agonist
of urokinase plasminogen activator comprising, culturing uPAR bearing
cells in the presence of lysine, or an analog of lysine, e.g.,
epsilon-amino caproic acid or tranexamic acid (trans-4-(amino methyl)
cyclohexane carboxylic acid, contacting the cells with a candidate
compound; and comparing the level of mitogenic activity, in the presence
of the candidate compound to the level of mitogenic activity in the
absence of the candidate compound, a higher level of activity in the
presence of the compound being indicative that the compound is an agonist.
In another aspect, the invention features a method of inhibiting the
interaction of urokinase plasminogen activator with urokinase plasminogen
activator receptor including contacting the receptor with lysine, or an
analog of lysine, e.g., epsilon-amino caproic acid or tranexamic acid
(trans-4-(amino methyl) cyclohexane carboxylic acid to inhibit the
interaction.
In another aspect, the invention features a method for treating an animal
having a disorder including identifying an animal at risk for the
disorder; and administering, e.g., topically, a therapeutically-effective
amount of lysine, or an analog of lysine, e.g., epsilon-amino caproic acid
or tranexamic acid (trans-4-(amino methyl) cyclohexane carboxylic acid to
the animal.
In preferred embodiments: the concentration, amount, or dosage of lysine or
an analog administered to the animal results in a concentration of lysine
or an analog thereof at the surface of a keratinocyete of the animal that
is greater than the highest concentration that can be achieved at the
surface of the cell by oral, intravenous, or other systemic administration
without deleterious effect on the animal; the concentration, amount, or
dosage of lysine or an analog thereof administered to the animal results
in a concentration of lysine or an analog thereof at the surface of a
keratinocyte of the animal greater than the highest concentration that is
achieved at the surface of the cell when lysine an analog thereof is
administered by oral, intravenous, or other systemic administration for
nutritional purposes; sufficient lysine is administered such that the
concentration of lysine in the dermis is higher, e.g., at least 20, 50,
80, 200, or 400% higher than the concentration of lysine in the dermis of
a normal individual or of an individual receiving intravenous nutrition.
In another aspect, the invention features a method for mitogenically
stimulating a cell, e.g., a uPAR bearing cell, e.g., a keratinocytic,
including contacting the cell with an effective amount of lysine, or an
analog of lysine, e.g., epsilon-amino caproic acid or tranexamic acid
(trans-4-(amino methyl) cyclohexane carboxylic acid.
In preferred embodiments: the lysine or analog thereof is provided at a
concentration greater than is provided in growth medium used to culture
said cells; lysine is provided at a concentration greater than found in
fetal calf or bovine serum.
In another aspect, the invention features a method of delivering a compound
to a uPAR bearing cell including providing a chimeric molecule which
includes the compound coupled to lysine, or an analog of lysine, e.g.,
epsilon-amino caproic acid or tranexamic acid (trans-4-(amino methyl)
cyclohexane carboxylic.
In another aspect, the invention features a method of inhibiting the
proteolytic destruction of an extracellular protein matrix which includes
cells bearing the uPAR including contacting cells bearing the uPAR with
lysine, or an analog of lysine, e.g., epsilon-amino caproic acid or
tranexamic acid (trans-4-(amino methyl) cyclohexane carboxylic acid.
In another aspect, the invention features therapeutic composition, e.g., a
therapeutic composition suitable for topical application, including as an
active ingredient lysine or an analog of lysine, e.g., epsilon-amino
caproic acid or tranexamic acid (trans-4-(amino methyl) cyclohexane
carboxylic acid, and a pharmaceutically-acceptable carrier. In preferred
embodiments the amount of lysine or an analog in the composition is
sufficient that one, two, three, five, or less than ten administrations of
the composition to the patient results in: a concentration of lysine or an
analog thereof at the surface of a keratinocyete of the patient that is
greater than the highest concentration that can be achieved at the surface
of the cell by oral, intravenous, or other systemic administration without
deleterious effect on the animal; a concentration of lysine or an analog
thereof at the surface of a keratinocyete of the patient greater than the
highest concentration that is achieved at the surface of the cell when
lysine an analog thereof is administered by oral, intravenous, or other
systemic administration for nutritional purposes.
Substantially pure or purified DNA is DNA that is not immediately
contiguous with both of the coding sequences with which it is immediately
contiguous (i.e., one at the 5' end and one at the 3' end) in the
naturally-occurring genome of the organism from which the DNA of the
invention is derived. The term therefore includes, for example, a
recombinant DNA which is incorporated into a vector; into an autonomously
replicating plasmid or virus; or into the genomic DNA of a prokaryote or
eukaryote, or which exists as a separate molecule (e.g., a cDNA or a
genomic DNA fragment produced by PCR or restriction endonuclease
treatment) independent of other DNA sequences. It also includes a
recombinant DNA which is part of a hybrid gene encoding additional
polypeptide sequence.
Homologous refers to the sequence similarity between two polypeptide
molecules or between two nucleic acid molecules. When a position in both
of the two compared sequences is occupied by the same base or amino acid
monomeric subunit, e.g., if a position in each of two DNA molecules is
occupied by adenine, then the molecules are homologous at that position.
The homology between two sequences is a function of the number of matching
or homologous positions shared by the two sequences. For example, 6 of 10,
of the positions in two sequences are matched or homologous then the two
sequences are 60% homologous. By way of example, the DNA sequences ATTGCC
and TATGGC share 50% homology.
A substantially pure or purified preparation of a peptide is a preparation
which is substantially free of the peptides or proteins with which the
peptide (or the protein from which it is derived, e.g., whole length uPA,
in the case of synthetic uPA peptides) naturally occurs in a cell.
The inventors have found that the ability to bind to uPAR and the mitogenic
activity of uPA is preserved with uPA peptides as small as a 9-mer (GFD
21-29) (i.e., uPA residues 21-29 of the GFD), whose sequence is
Ser-Asn-Lys-Tyr-Phe-Ser-Asn-Ile-His ((SEQ ID NO:1), and a 6-mer (GFD
21-26), whose sequence is Ser-Asn-Lys-Tyr-Phe-Ser (SEQ ID NO:2). These
peptides can stimulate the growth of keratinocytes without causing side
effects of tissue bleeding. The invention allows for promoting the repair
of injured tissue by causing epidermal cells to be stimulated to grow and
thus repair the wound.
The small size of uPA peptides of the invention, e.g., the 9-mer and the
6-mer, is important in that smaller peptides will more readily penetrate
to the basal layer (the layer with mitotoic activity in the normal
epidermis) and are more effective in topically stimulating epidermal cells
to grow.
Peptides and methods of the invention can be used to stimulate the growth
of epidermal sheets of cells in vitro or in vivo. These sheets of cells
can be used, e.g., as covering for large areas of denuded skin, such as
bums. The invention provides a method for stimulation of the epidermal
cells, not only to produce epidermal sheets more rapidly and effectively,
but also to stimulate continued growth of the epidermal cells once they
are place upon the wounded tissue. The invention will enhance the ability
of in vitro grown epidermal sheets applied to a patient to further grow
and produce basement membrane components for effective attachment to the
underlying dermis.
uPA peptides of the invention are also useful for blocking the binding of
uPA to the uPAR, to thus inhibit the activity of uPA. uPA peptides having
mitogenic activity, uPA peptides which bind uPAR but which do not
stimulate mitogenic activity, or uPA peptides which inhibit mitogenisis,
can be used. Peptides having mitogenic activity will bind and allow for
cell proliferation without the proteolytic effects of uPA. The peptides
not having mitogenic activity will block uPA but exhibit neither mitogenic
activity nor proteolytic activity.
The inventors have also discovered that the amino acid at residue 23 of uPA
plays an important role in the activity of a uPA peptide. As is described
below, uPA peptides with enhanced mitogenic activity and uPA peptides with
inhibitory properties can be synthesized by replacing the lysine normally
found at position 23. Substituted peptides are useful for inhibiting or
enhancing the growth or proliferation of cells and for receptor binding
studies.
Other features and advantages of the invention will be apparent from the
following description and from the claims.
DETAILED DESCRIPTION
The drawings will first be briefly described.
Drawings
FIG. 1 is a bar graph which represents the effect of various uPA peptides
on DNA synthesis in keratinocytes. Various concentrations of GFD peptides
were tested in order to compare the mitogenic activity. After 24 h
incubation with a peptide and 3H-thymidine, incorporated radioactivity was
measured as described herein. Five concentrations were used for each
peptide. For a given peptide the uppermost bar of the five represents 0.1
.mu.M, the next lower bar 0.33 .mu.M, the next lower bar 1 .mu.M, the next
lower bar 3.3 .mu.M, and the lowest bar, 10 .mu.M.
FIG. 2 is a bar graph which represents the additive effect of the 6-mer
peptide in different growth media. XBM, keratinocyte basal medium
(keratinocyte growth medium without epidermal growth factor, insulin,
hydrocortisone, or bovine pituitary extract); KGM-BPE, keratinocyte growth
medium without bovine pituitary extract; BM+Ins, keratinocyte basal medium
plus insulin; BM+EGF, keratinocyte basal medium plus epidermal growth
factor; BM+BPE, keratinocyte basal medium plus bovine pituitary extract;
KGM, keratinocyte growth medium (Clonetics, Calif.).
FIG. 3 is a graphic representation of uPA activity in keratinocyte culture
over a 22 day period. The solid line represents total cell number; the
dotted line represents total uPA activity in the medium; and the dashed
line represents the uPA activity per cell.
Mitogenic Activity of uPA Fragments in Keratinocytes
uPA is a 55 Kda protein which has an EGF-like domain, a kringle domain, and
a trypsin-like protease domain (uPA has been cloned, see Riccio et al.,
1985, Nucleic Acids Research 13: No. 8). uPA binds to a specific cell
receptor (uPAR) through the EGF-like domain (uPAR has been cloned, see
Roldan, et al., 1990 EMBO Journal 9:467). The 18 Kda amino terminal region
of urokinase which includes the EGF-like domain and the kringle domain has
been found to induce mitogenesis in an osteoblast cell line (Rablani et
al., Biochem. Biophys. Res. Commun. 173:1058,1990).
In order to obtain small molecules which retained the mitogenic activity of
uPA, but which did not have fibrinolytic activity, a number of peptides of
various lengths were designed to determine the portion or portions of the
uPA molecule which is required for mitogenic activity. The synthesis and
analysis of these peptides is described below.
Preparation of Peptides Synthetic peptides of various lengths were
synthesized according to standard methods using a peptide synthesizer
(Research Genetics, Inc.). Peptides were purified by reverse phase HPLC
(Toso, Inc.). The amino acid sequences are given in Table 1.
Mitogenesis Assay The effect of a peptide on mitogenesis was determined as
a function of DNA synthesis as follows. Peptides (in phosphate buffered
saline (PBS)) were added to the monolayers (grown as described herein) to
a final concentration of either 0.1 .mu.M, 0.33 .mu.M, 1.0 .mu.M, 3.3
.mu.M, or 10 .mu.M. 5 .mu.L of .sup.3 H-thymidine (10 .mu.Ci/ml; Amersham,
Arlington Heights, Ill.) in 150 .mu.L KGM was added to the cultures
concurrent with the peptides, and cells were harvested and lysed after 24
hours at 37.degree. C. in distilled H.sub.2 O using a Tomtec cell
harvester, and fixed with 70% ethanol. The lysed cells were then
transferred to a nylon filter (Amersham, Arlington Heights, Ill.), Beta
plate scintillation fluid was added (LKB), and radioactivity was measured
using a Wallac 1205 beta plate counter. Control samples, incubated in the
presence of phosphate-buffered saline (PBS) and .sup.3 H-thymidine gave
control levels of radioactivity of approximately 400 cpm.
Cell Culture Primary human keratinocytes (Clonetics, Calif.) were cultured
in 96-well plates (Corning, N.Y.) in serum-free medium (KGM medium,
Clonetics) to 70-80% confluency on a monolayer culture at 37.degree. C. in
5% C02.
Short uPA Peptides Have Mitogenic Activity Several short uPA peptides were
synthesized and tested for mitogenic activity as described herein. The
peptides are shown in Table 1.
TABLE 1
______________________________________
Sequence of uPA Peptides
______________________________________
21-mer: DCLNGGTCVSNKYFSNIHWCN
(Seq ID No 3)
18-mer: NGGTCVSNKYFSNIHWCN (Seq ID No 4)
15-mer: TCVSNKYFSNIHWCN (Seq ID No 5)
12-mer: TCVSNKYFSNIH (Seq ID No 6)
9-mer: SNKYFSNIH (Seq ID No 1)
6-mer: SNKYFS (Seq ID No 2)
______________________________________
As shown in FIG. 1, all of the peptides tested stimulated thymidine
incorporation. The 6-mer peptide did not demonstrate any measurable
activity at concentrations up to 3.3 .mu.M, but at a concentration of
10.mu.M resulted in a significant stimulation of .sup.3 H-thymidine
incorporation. The 9-mer peptide was effective in stimulating mitogenesis
at concentrations as low as 0.33 .mu.M, with a maximum level of mitogenic
activity at 1 .mu.M. The 12-mer demonstrated mitogenic activity only at 1
.mu.M, and had little, if any, effect on keratinocyte stimulation at other
concentrations. The highest levels of mitogenic activity produced by the 1
5-mer occurred at a concentration of 3.3 .mu.M. At a concentration of 3.3
.mu.M and 10 .mu.M, the 21 -mer and the 18-mer showed similar effects on
the stimulation of mitogenesis in keratinocytes. PBS controls showed
approximately 400 cpm of incorporation. The entire AFF was also found to
stimulate keratinocyte mitogenesis.
The Interaction of the 6-mer peptide (GFD 21-26) with Other Growth Factors
The 6-mer peptide (GFD 21-26), was studied further in order to determine
its effect on keratinocyte stimulation in the presence or absence of other
factors. Keratinocytes were cultured in various growth media which lacked
different growth factors which are normally present in KGM (complete
medium) for 24 hours in the presence or absence of the 6-mer (FIG. 2), and
.sup.3 H-thymidine incorporation was measured as described above.
As shown in FIG. 2, the 6-mer (P-8) stimulated .sup.3 H-thymidine uptake
greatly in the presence of the complete keratinocyte growth medium which
contains various growth factors such as bovine pituitary extract (BPE),
insulin, EGF, and hydrocortisone. In addition, the 6-Mer always showed
additive effect to any growth factors tested in this study.
Expression of Urokinase Plasminogen Activator Receptor (uPAR) on
Proliferating Keratinocytes
Northern blot analysis was utilized to determine the level of uPAR
expression in keratinocytes at various stages of growth. Briefly, a 0.4 kb
probe which encodes the ectodomain of the uPAR was prepared by the RT-PCR
technique (Invitrogen, cDNA cycle kit) using the primers:
5'-GGGGATTGCCGTGTGGAAGA-3'(SEQ ID NO:7) and
5'-GGAATTCGAAGGTAGCCACAGCCACGGAG-3' (SEQ ID NO:8). Messenger RNA was
purified from cultured keratinocytes in monolayer culture at the stages of
50% confluency, 80% confluency, 100% confluency, and after confluency
(120% confluency). Approximately 2 1.mu.g of purified mRNA from each
cultured keratinocyte stage was then subjected to electrophoresis in
agarose and transferred to a nylon membrane (Hybond N). After
pre-hybridization in 6.times.SSC, 5.times.Denhardt's, 0.5% SDS, 100
.mu./ml sonicated salmon sperm DNA, and 50% deionized formamide, the
[.sup.32 P]-labeled uPAR probe was added, and hybridization was allowed to
proceed at 42.degree. C. overnight in the presence of 50% formamide. The
nylon membrane was then washed twice with 2.times.SSC containing 0.1% SDS,
followed by washes in 0.1.times.SSC, 0.1% SDS at 37.degree. C. for 30 mins
and then at 55.degree. C. for 30 mins. Autoradiography was performed by
exposing Kodak XRR film at-70.degree. C.
The data of the Northern blot analysis indicated that uPAR mRNA is highly
expressed during stages of exponential growth (e.g., at 50% confluency).
In addition, the data also indicate that the expression of uPAR mRNA
decreases as confluency increases over 50%, and is almost undetectable at
100% confluency. These results indicate that there is a direct correlation
between the expression of the UPAR mRNA and keratinocyte proliferation.
Urokinase Plasminogen Activator Activity in Keratinocytes
Normal human keratinocytes (Clonetics, Calif.) were cultured in 96-well
plates using serum free keratinocyte growth medium and cell number was
monitored every two days by hemocytometer. uPA activity was also measured
every two days using a two-step assay. Briefly, 50 .mu.1 of conditioned
medium from the keratinocyte culture was mixed with 0.1 M Tris-HCl, pH
8.5, containing 0.1% Tween-20, and 50 .mu.l 0.1 mg/ml purified plasminogen
in PBS, and incubated for 30 mins at 30.degree. C. Fifty microliters of
0.2 M phosphate buffer, pH 7.2, containing 1.4 M NaCl mixed with 4 mM of
the substrate S-2251 (Val-Leu-Lys-p-nitroanilide; Kabi Diagnostics,
Stockholm) to a final concentration of 0.8 mM S-2251, was then added.
After incubation, the absorbence of each sample was measured at 405 nm,
and uPA activity was determined using the International Standard of
urokinase.
FIG. 3 summarizes the uPA activity in normal human epithelial
keratinocytes. The unbroken line represents the cell number of
keratinocytes in culture over a 22 day period, and shows that cell number
rapidly increases from day 6, approaches 100% confluency at day 8, then
continues to increase after 100% confluency at day 12, and begins to
decline at approximately day 14. The dotted line represents the uPA
activity over the same 22 day period. Total uPA production increases up to
approximately day 12 and then decreases. The dashed line represents the
uPA activity per cell and demonstrates that uPA activity per cell
increases prior to confluence, and then decreases when confluence is
reached. These data further substantiate that there is a direct
correlation between uPA activity and keratinocyte proliferation.
uPA Peptides with Altered Activities
Computer analysis of the structure of uPA regions suggested the presence of
a turn-structure at Lys.sup.23 of GFD. The effect of substitutions at
Lys.sup.23 was evaluated in uPA peptides. 9mers were synthesized wherein
Lys.sup.23 was replaced by: alanine, Ser-Asn-Ala-Tyr-Phe-Ser-Asp-Ile-His
(SEQ ID NO:20) (A-GFD); arginine, Ser-Asn-Arg-Tyr-Phe-Ser-Asp-Ile-His (SEQ
ID NO:21) (R-GFD); and glutamic acid, Ser-Asn-Glu-Tyr-Phe-Ser-Asp-Ile-His
(SEQ ID NO:22) (E-GFD).
The ability of these substituted peptides to stimulate mitogenesis was
determined by .sup.3 H-thymidine uptake. As expected the positive control
9-mer Ser-Asn-Lys-Tyr-Phe-Ser-Asp-Ile-His (SEQ ID NO:1) (K-GFD) increased
uptake as compared with untreated cells. .sup.3 H-thymidine uptake in
cells treated with A-GFD was little different from negative control cells
(no peptide). R-GFD greatly increased .sup.3 H-thymidine uptake, as
compared with K-GFD-treated positive control cells. E-GFD on the other
hand inhibited mitogenesis, .sup.3 H-thymidine uptake was less than that
seen in negative contact cells (no peptide added). These results reveal
that the amino acid residue at position 23 plays an important role in
keratinocyte stimulation. Furthermore, the activity is dependent on charge
as the move to a more basic residue enhanced mitogenic activity whereas
substitution with negatively charged Glu resulted in an inhibitory effect.
Other useful peptides can be found by synthesizing uPA peptides, e.g.,
peptides shown in Table 1 with various substitutions at position 23 (or
other positions) and determining their effect on mitogenesis.
Use
The formulations of this invention are especially useful for topical
administration, but may also be administered in other modes, e.g.,
parenterally, intravenously, subcutaneously, or intramuscularly.
Therapeutically effective amounts (e.g., amounts which eliminate or reduce
the patient's pathological condition) of the peptides of the invention can
be administered to humans or other mammals to treat or inhibit conditions
or disorders wherein inhibition or stimulation of cell growth is desired,
e.g., in disorders wherein the promotion of keratinocyte growth is
desirable.
The compounds provided herein can be formulated into pharmaceutical
compositions by admixture with any pharmaceutically acceptable nontoxic
excipients and carriers. As noted above, such compositions may be prepared
for parenteral administration, particularly in the form of liquid
solutions or suspensions; for oral administration, particularly in the
form of tablets or capsules; or topically in the form of ointments, creams
or gels. The compositions may conveniently be administered in unit dosage
form and may be prepared by any of the methods well known in the
pharmaceutical art. Formulations for parenteral administration may contain
as common excipients, sterile water or saline, polyalkylene glycols such
as polyethylene glycol, oils of vegetable origin, hydrogenated
naphthalenes and the like. In particular, biocompatible, biodegradable
lactide polymer, lactide/glycoside copolymer, or
polyoxyethylene-polyoxypropylene copolymers may be useful excipients to
control the release of the peptides. Other potentially useful parenteral
delivery systems for these peptides include ethylene-vinyl acetate
copolymer particles, osmotic pumps, implantable infusion systems, and
liposomes. Formulations for parenteral administrations may also include
glycocholate for buccal administration, methoxysalicylate for rectal
administration, or citric acid for vaginal administration.
The materials of this invention can be employed as the sole active agent in
a pharmaceutical composition or can be used in combination with other
active ingredients, e.g., other compounds which facilitate cell growth or
inhibition, or peptidase or protease inhibitors.
The dosage of the compounds described herein in a therapeutic composition
will vary depending upon a number of factors, including the route of
administration, type and state of the disease, and the overall health
status of the particular patient.
The peptides of the invention can also be used in vitro, e.g., to stimulate
the growth of cultured epidermal cells.
Other Embodiments
The invention includes any peptide which is substantially homologous to a
uPA peptide described herein and which has biological activity. By
"biologically active" is meant the ability to bind specifically to a uPAR
bearing cell, e.g., a keratinocyte, or the ability to promote or inhibit
the growth (mitogenesis) of a uPAR bearing cell, e.g., a keratinocyte, as
determined by the assays described herein or by other assays know to those
in the art. Most preferably substantially homologous peptides, fragments
or analogs will have: 10%, preferably 40%, more preferably at least 90,
95, or 99%, of the activity of the 9-mer of Table 1 in the case of growth
promoting peptides; and 10%, preferably 40%, more preferably at least 90,
95, or 99%, of the inhibitory activity of E-GFD, in the case of inhibitory
peptides. Peptides which bind but have no effect on mitogenesis have
biological activity if they bind at least 10, preferably 40, or more
preferably at least 90, 95, or 99%, as well as the peptide of SEQ ID NO:1.
The invention also includes chimeric peptides that include uPA peptides
described herein.
The invention also includes any biologically active fragment or analog of
the uPA peptides described herein. Preferred analogs include peptides
whose sequences differ from the wild-type sequence (i.e., the sequence of
the homologous portion of naturally occurring uPA) only by conservative
amino acid substitutions, preferably by only one, two, or three,
substitutions, for example, substitution of one amino acid for another
with similar characteristics (e.g., valine for glycine, arginine for
lysine, etc.) or by one or more non-conservative amino acid substitutions,
deletions, or insertions which do not abolish the polypeptide's biological
activity. Table 2 lists a number of conservative amino acid substitutions.
TABLE 2
______________________________________
CONSERVATIVE AMINO ACID REPLACEMENTS
For Amino Acid
Code Replace With
______________________________________
Alanine A D-Ala, Gly, Aib, .beta.-Ala, Acp, L-Cys, D-Cys
Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg
Met, Ile, D-Met, D-Ile, Orn, D-Orn
Asparagine
N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln
Aspartic Acid
D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln
Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr
Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp
Glutamic Acid
E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln
Glycine G Ala, D-Ala, Pro, D-Pro, Aib, .beta.-Ala, Acp
Isoleucine
I D-Ile, Val, D-Val, AdaA, AdaG, Leu, D-Leu,
Met, D-Met
Leucine L D-Leu, Val, D-Val, AdaA , AdaG, Leu, D-Leu,
Met, D-Met
Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg,
Met, D-Met, Ile, D-Ile, Orn, D-Orn
Methionine
M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu,
Val, D-Val
Phenylalanine
F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His,
Trp, D-Trp, Trans-3,4, or 5-phenylproline,
AdaA, AdaG, cis-3,4, or 5-phenylproline,
Bpa, D-Bpa
Proline P D-Pro, L-I-thioazolidine-4-carboxylic
acid, D-or L-1-oxazo1idine-4-carboxylic
acid (Kauer, U.S. Pat. No. 4,511,390)
Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met,
Met(O), D-Met(O), L-Cys, D-Cys
Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met,
Met(O), D-Met(O), Val, D-Val
Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, D-His
Valine V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met,
AdaA, AdaG
______________________________________
Other useful modifications include those which increase peptide stability;
such analogs may contain, for example, one or more non-peptide bonds
(which replace the peptide bonds) or D-amino acids in the peptide
sequence.
Analogs can differ from naturally occurring uPA sequence in amino acid
sequence or can be modified in ways that do not involve sequence, or both.
Analogs of the invention will generally exhibit at least 40%, more
preferably 50%, more preferably 60%, more preferably 70%, more preferably
80%, more preferably 90%, and most preferably 95% or even 99%, homology
with a naturally occurring uPA sequence or with a uPA sequence described
herein.
Non-sequence modifications include in vivo or in vitro chemical
derivatization of peptides, e.g., acetylation, methylation,
phosphorylation, carboxylation, or glycosylation. Also included are
analogs that include residues other than naturally occurring L-amino
acids, e.g., D-amino acids or non-naturally occurring or synthetic amino
acids, e.g., .beta. or .gamma. amino acids. Alternatively, increased
stability may be conferred by cyclizing the peptide molecule.
Analogs, e.g., peptides differing by 1,2,3, or more residues from the
peptides disclosed herein, can be prepared by methods known to those in
the art and tested for biological activity by methods known in the art or
disclosed herein.
Other embodiments are within the following claims.
__________________________________________________________________________
# SEQUENCE LISTING
- (1) GENERAL INFORMATION:
- (iii) NUMBER OF SEQUENCES: 25
- (2) INFORMATION FOR SEQ ID NO:1:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 9 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
- Ser Asn Lys Tyr Phe Ser Asn Ile His
1 5
- (2) INFORMATION FOR SEQ ID NO:2:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 6 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
- Ser Asn Lys Tyr Phe Ser
1 5
- (2) INFORMATION FOR SEQ ID NO:3:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 21 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
- Asp Cys Leu Asn Gly Gly Thr Cys Val Ser As - #n Lys Tyr Phe Ser Asn
# 15
- Ile His Trp Cys Asn
20
- (2) INFORMATION FOR SEQ ID NO:4:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 18 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
- Asn Gly Gly Thr Cys Val Ser Asn Lys Tyr Ph - #e Ser Asn Ile His Trp
# 15
- Cys Asn
- (2) INFORMATION FOR SEQ ID NO:5:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 15 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
- Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn Il - #e His Trp Cys Asn
# 15
- (2) INFORMATION FOR SEQ ID NO:6:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 12 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
- Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn Il - #e His
# 10
- (2) INFORMATION FOR SEQ ID NO:7:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 20 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: cDNA
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
# 20 AAGA
- (2) INFORMATION FOR SEQ ID NO:8:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 29 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: cDNA
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
# 29 CACA GCCACGGAG
- (2) INFORMATION FOR SEQ ID NO:9:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 10 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
- Asp Cys Leu Asn Gly Gly Thr Cys Val Ser
# 10
- (2) INFORMATION FOR SEQ ID NO:10:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 9 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
- Cys Leu Asn Gly Gly Thr Cys Val Ser
1 5
- (2) INFORMATION FOR SEQ ID NO:11:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 8 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
- Leu Asn Gly Gly Thr Cys Val Ser
1 5
- (2) INFORMATION FOR SEQ ID NO:12:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 7 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
- Asn Gly Gly Thr Cys Val Ser
1 5
- (2) INFORMATION FOR SEQ ID NO:13:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 6 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
- Gly Gly Thr Cys Val Ser
1 5
- (2) INFORMATION FOR SEQ ID NO:14:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 5 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
- Gly Thr Cys Val Ser
1 5
- (2) INFORMATION FOR SEQ ID NO:15:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 4 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
- Thr Cys Val Ser
- (2) INFORMATION FOR SEQ ID NO:16:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 4 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
- Ser Asn Ile His
1
- (2) INFORMATION FOR SEQ ID NO:17:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 5 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
- Ser Asn Ile His Trp
1 5
- (2) INFORMATION FOR SEQ ID NO:18:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 6 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
- Ser Asn Ile His Trp Cys
1 5
- (2) INFORMATION FOR SEQ ID NO:19:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 7 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
- Ser Asn Ile His Trp Cys Asn
1 5
- (2) INFORMATION FOR SEQ ID NO:20:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 9 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
- Ser Asn Ala Tyr Phe Ser Asp Ile His
1 5
- (2) INFORMATION FOR SEQ ID NO:21:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 9 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
- Ser Asn Arg Tyr Phe Ser Asp Ile His
1 5
- (2) INFORMATION FOR SEQ ID NO:22:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 9 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
- Ser Asn Glu Tyr Phe Ser Asp Ile His
1 5
- (2) INFORMATION FOR SEQ ID NO:23:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 20 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
- Leu Asn Gly Gly Thr Cys Val Ser Asn Lys Ty - #r Phe Ser Asn Ile His
# 15
- Trp Cys Asn Cys
20
- (2) INFORMATION FOR SEQ ID NO:24:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 18 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
- Gly Gly Thr Cys Val Ser Asn Lys Tyr Phe Se - #r Asn Ile His Trp Cys
# 15
- Asn Cys
- (2) INFORMATION FOR SEQ ID NO:25:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 157 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v) FRAGMENT TYPE: internal
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
- Ser Asn Glu Leu His Gln Val Pro Ser Asn Cy - #s Asp Cys Leu Asn Gly
# 15
- Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser As - #n Ile His Trp Cys Asn
# 30
- Cys Pro Lys Lys Phe Gly Gly Gln His Cys Gl - #u Ile Asp Lys Ser Lys
# 45
- Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Ar - #g Gly Lys Ala Ser Thr
# 60
- Asp Thr Met Gly Arg Pro Cys Leu Pro Trp As - #n Ser Ala Tyr Val Leu
#80
- Gln Gln Thr Tyr His Ala His Arg Ser Gln Al - #a Leu Gln Leu Gly Leu
# 95
- Gly Lys His Asn Tyr Cys Arg Asn Pro Gln As - #n Arg Arg Arg Pro Trp
# 110
- Cys Tyr Tyr Gln Val Gly Leu Lys Pro Leu Va - #l Gln Glu Cys Met Val
# 125
- His Asp Cys Ala Asp Gly Lys Lys Pro Ser Se - #r Pro Pro Glu Glu Leu
# 140
- Lys Phe Gln Cys Gly Gln Lys Tyr Leu Arg Pr - #o Arg Phe
145 1 - #50 1 - #55
__________________________________________________________________________
Top